Biomedical subjects

P Steinecke

Publications and source records attributed to P Steinecke.

5 recordsLinked to original sources

PubMed1998

Early events of tomato spotted wilt transcription and replication in protoplasts.

A protoplast transfection system for tomato spotted wilt tospovirus (TSWV) was established by PEG-mediated infection of tobacco protoplasts. Analysis of viral RNA synthesis revealed an asymmetric production of viral (v) and viral-complementary (vc) strands of all three genomic RNA segments and a separation in time of subgenomic transcription during the first 72 hr post infection (p.i.). Synthesis of vc-RNA was detectable 8-10 hrs prior to v-RNA production. During the first 72 h of infection the accumulation of S-RNAs exceeded M- and L-RNAs and the amount of vc-RNAs was larger than that of genomic v-RNAs. The subgenomic N-mRNA was first detected 5 hr p.i., followed by vc-S RNA and the mRNA for the nonstructural protein (NSs) 15 hr later. The two subgenomic mRNA species of the S RNA appeared to be regulated independently from each other and from other viral mRNA species. Defective interfering (DI) elements from L RNA associated with the virus inoculum, were not preferentially replicated at the expense of full-sized L-RNA.

Immunohistochemistry↗

PubMed1995

Ribozymes.

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Base Sequence↗

PubMed1994

Expression of a reporter gene is reduced by a ribozyme in transgenic plants.

A chimeric gene encoding a ribozyme under the control of the cauliflower mosaic virus (CaMV) 35S promoter was introduced into transgenic tobacco plants. In vivo activity of this ribozyme, which was designed to cleave npt mRNA, was previously demonstrated by transient expression assays in plant protoplasts. The ribozyme gene was transferred into transgenic tobacco plants expressing an rbcS-npt chimeric gene as an indicator. Five double transformants out of sixteen exhibited a reduction in the amount of active NPT enzyme. To measure the amount of ribozyme produced, in the absence of its target, the ribozyme and target genes were separated by genetic segregation. The steady-state concentrations of ribozyme and target RNA were shown to be similar in the resulting single transformants. Direct evidence for a correlation between reduced npt gene expression and ribozyme expression was provided by crossing a plant containing only the ribozyme gene with a transgenic plant expressing the npt gene under control of the 35S promoter, i.e. the same promoter used to direct ribozyme expression. The expression of npt was reduced in all progeny containing both transgenes. Both steady-state levels of npt mRNA and amounts of active NPT enzyme are decreased. In addition, our data indicate that, at least in stable transformants, a large excess of ribozyme over target is not a prerequisite for achieving a significant reduction in target gene expression.

Drug Resistance↗

PubMed1994

A stable hammerhead structure is not required for endonucleolytic activity of a ribozyme in vivo.

Cleavage of a specific target, the mRNA encoding the bacterial neomycin phosphotransferase, by mutant satellite RNA of subterranean clover mottle virus (sSCMoV) ribozymes (Rz) was used to study the role of the hammerhead (Hh) structure in Rz activity in cis and in trans. The bimolecular Rz-target RNA interaction was predicted by computer secondary structure analysis. In vivo, endonucleolytic cleavage was determined in plant protoplasts and compared with in vitro results. Two point mutations within the Hh were studied in detail. A Rz mutant with a point mutation in the most distal nucleotide of the catalytic domain (A14G) showed no endonucleolytic activity in vivo. A second point mutation inside helix II (G11.3C) which destabilizes the helix and, according to thermodynamic calculations, should disrupt the conserved Hh structure, unexpectedly displayed Rz activity in trans in vivo. In vitro, this mutant exhibited an activity similar to the wild-type Rz in cis, but no significant activity in trans. It therefore appears that helix II within the Rz Hh structure is not required in vivo for endonucleolytic activity, nor for stability of the Rz transcript, and that in vitro results are inadequate to predict Rz activity in living cells.

Amino Acid Sequence↗

PubMed1992

Expression of a chimeric ribozyme gene results in endonucleolytic cleavage of target mRNA and a concomitant reduction of gene expression in vivo.

The subclass of catalytic RNAs termed ribozymes cleave specific target RNA sequences in vitro. Only circumstantial evidence supports the idea that ribozymes may also act in vivo. In this study, ribozymes with a hammerhead motif directed against a target sequence within the mRNA of the neomycin phosphotransferase gene (npt) were embedded into a functional chimeric gene. Two genes, one containing the ribozyme and the other producing the target, were cotransfected into plant protoplasts. Following in vivo expression, a predefined cleavage product of the target mRNA was detected by ribonuclease protection. Expression of both the ribozyme gene and the target gene was driven by the CaMV 35S promoter. Concomitant with the endonucleolytic cleavage of the target mRNA, a complete reduction of NPT activity was observed. An A to G substitution within the ribozyme domain completely inactivates ribozyme-mediated hydrolysis but still shows a reduction in NPT activity, albeit less pronounced. Therefore, the reduction of NPT activity produced by the active ribozyme is best explained by both hydrolytic cleavage and an antisense effect. However, the mutant ribozyme--target complex was more stable than the wildtype ribozyme--target complex. This may result in an overestimation of the antisense effect contributing to the overall reduction of gene expression.

Amino Acid Sequence↗